Septin‐based readout of PI(4,5)P2 incorporation into membranes of giant unilamellar vesicles

International audienceSeptins constitute a novel class of cytoskeletal proteins. Budding yeast septins self-assemble into non-polar filaments bound to the inner plasma membrane through specific interactions with L-α-phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2). Biomimetic in vitro assays using Giant Unilamellar Vesicles (GUVs) are relevant tools to dissect and reveal insights in proteins-lipids interactions, membrane mechanics and curvature sensitivity. GUVs doped with PI(4,5)P2 are challenging to prepare. This report is dedicated to optimize the incorporation of PI(4,5)P2 lipids into GUVs by probing the proteins-PI(4,5)P2 GUVs interactions. We show that the interaction between budding yeast septins and PI(4,5)P2 is more specific than using usual reporters (phospholipase C1). Septins have thus been chosen as reporters to probe the proper incorporation of PI(4,5)P2 into giant vesicles. We have shown that electro-formation on platinum wires is the most appropriate method to achieve an optimal septin-lipid interaction resulting from an optimal PI(4,5)P2 incorporation for which, we have optimized the growth conditions. Finally, we have shown that PI(4,5)P2 GUVs have to be used within a few hours after their preparation. Indeed, over time, PI(4,5)P2 is expelled from the GUV membrane and the PI(4,5)P2 concentration in the bilayer decreases

Author Correction:Human ESCRT-III polymers assemble on positively curved membranes and induce helical membrane tube formation

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Propriétés mécaniques et fonction de CHMP2B dans la voie de remodelage et de scission membranaire par les ESCRT

The ESCRT-III protein complex mediates membrane remodeling in many cellular contexts. The ESCRT pathway has been extensively studied in vivo and partially reconstituted in vitro using yeast proteins. In Homo Sapiens, at least 12 ESCRT-III proteins exist, called Charged Multivesicular Body Protein (CHMP 1-7). Although, the main function of the ESCRT-III protein assemblies is to induce membrane scission by constricting membrane necks, the biophysical mechanism remains unclear and the mechanical properties of the CHMP polymers still poorly characterized. Moreover, the usually accepted sequence of recruitment of the major ESCRT components to the membrane prior to scission is CHMP4-CHMP3-CHMP2A but mammalian cells also have CHMP2B considered to be a CHMP2A isoform; so far, its role remains elusive. We have used biomimetic model systems and purified CHMP proteins to study in vitro protein affinity and effects on membrane by several techniques. We established that CHMP2B binding is enhanced with PI(4,5)P2 lipids, whereas the other human core components have no lipid specificity besides their negative charge. We showed that in the presence of CHMP2B, membranes become rigidified in contrast to CMHP2A as well as CHMP4 and CHMP3, suggesting that CHMP2A and CHMP2B have very distinctive properties. Finally, we show in disagreement with the proposed models, that CHMP4 alone cannot deform membranes. In fact, it requires the interaction with CHMP2B or CHMP2A+3 proteins to do so, forming polymer assemblies that stabilizes tubular membrane structures. These observations provide a novel basis for proposing possible mechanism for membrane constriction in the presence of the ATPase Vps4.Le complexe protéique ESCRT-III conduit au remodelage membranaire dans de nombreux contextes cellulaires. Les ESCRTs ont été largement étudiés in vivo et partiellement reconstitués in vitro sur des protéines de levure. Chez l’Homo Sapiens, il existe au moins 12 protéines ESCRT-III appelées Charged Multivesicular Body Protein (CHMP 1-7). Malgré que la fonction principale de ces assemblages de protéines soit d’induire la scission membrane par constriction du cou des membranes, le mécanisme biophysique reste incertain et les propriétés mécaniques des polymères CHMPs faiblement caractérisées. Par ailleurs, la séquence de recrutement à la membrane généralement considérée pour les composantes principales est CHMP4-CHMP3-CHMP2A. Mais, les cellules mammifères incluent aussi CHMP2B, considérée comme isoforme de CHMP2A et, dont le rôle est méconnu. Nous avons utilisé des systèmes biomimétiques de membranes modèles et des protéines CHMPs purifiées pour étudier in vitro leur affinité et leurs effets sur la membrane par différentes techniques. Nous avons établi que seule CHMP2B a une affinité spécifique aux lipides PI(4,5)P2. Nous montrons qu’en présence de CHMP2B, les membranes deviennent rigides en contraste avec les autres CHMPs et suggérant que CHMP2A et CHMP2B ont des propriétés distinctes. En fin, nous montrons en désaccord avec les modèles proposés, que CHMP4 seule ne peut pas déformer les membranes. Elle requiert l’interaction avec CHMP2B ou CHMP2A+3 pour former des assemblages de polymères qui stabilisent des structures tubulaires de la membrane. Ces observations offrent une nouvelle base pour proposer un mécanisme possible pour la constriction membranaire avec l’ATPase Vps4

Dynamic and Sequential Protein Reconstitution on Negatively Curved Membranes by Giant Vesicles Fusion

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The ESCRT protein CHMP2B acts as a diffusion barrier on reconstituted membrane necks

International audienceEndosomal sorting complexes required for transport (ESCRT)-III family proteins catalyze membrane remodeling processes that stabilize and constrict membrane structures. It has been proposed that stable ESCRT-III complexes containing CHMP2B could establish diffusion barriers at the post-synaptic spine neck. In order to better understand this process, we developed a novel method based on fusion of giant unilamellar vesicles to reconstitute ESCRT-III proteins inside GUVs, from which membrane nanotubes are pulled. The new assay ensures that ESCRT-III proteins polymerize only when they become exposed to physiologically relevant membrane topology mimicking the complex geometry of post-synaptic spines. We establish that CHMP2B, both full-length and with a C-terminal deletion (ΔC), preferentially binds to membranes containing phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Moreover, we show that CHMP2B preferentially accumulates at the neck of membrane nanotubes, and provide evidence that CHMP2B-ΔC prevents the diffusion of PI(4,5)P2 lipids and membrane-bound proteins across the tube neck. This indicates that CHMP2B polymers formed at a membrane neck may function as a diffusion barrier, highlighting a potential important function of CHMP2B in maintaining synaptic spine structures

The ESCRT-III isoforms CHMP2A and CHMP2B display different effects on membranes upon polymerization

International audienceBackground: ESCRT-III proteins are involved in many membrane remodeling processes including multivesicular body biogenesis as first discovered in yeast. In humans, ESCRT-III CHMP2 exists as two isoforms, CHMP2A and CHMP2B, but their physical characteristics have not been compared yet.Results: Here, we use a combination of techniques on biomimetic systems and purified proteins to study their affinity and effects on membranes. We establish that CHMP2B binding is enhanced in the presence of PI(4,5)P2 lipids. In contrast, CHMP2A does not display lipid specificity and requires CHMP3 for binding significantly to membranes. On the micrometer scale and at moderate bulk concentrations, CHMP2B forms a reticular structure on membranes whereas CHMP2A (+CHMP3) binds homogeneously. Thus, CHMP2A and CHMP2B unexpectedly induce different mechanical effects to membranes: CHMP2B strongly rigidifies them while CHMP2A (+CHMP3) has no significant effect.Conclusions: We therefore conclude that CHMP2B and CHMP2A exhibit different mechanical properties and might thus contribute differently to the diverse ESCRT-III-catalyzed membrane remodeling processes

Human ESCRT-III polymers assemble on positively curved membranes and induce helical membrane tube formation

International audienceEndosomal sorting complexes for transport-III (ESCRT-III) assemble in vivo onto membranes with negative Gaussian curvature. How membrane shape influences ESCRT-III polymerization and how ESCRT-III shapes membranes is yet unclear. Human core ESCRT-III proteins, CHMP4B, CHMP2A, CHMP2B and CHMP3 are used to address this issue in vitro by combining membrane nanotube pulling experiments, cryo-electron tomography and AFM. We show that CHMP4B filaments preferentially bind to flat membranes or to tubes with positive mean curvature. Both CHMP2B and CHMP2A/CHMP3 assemble on positively curved membrane tubes. Combinations of CHMP4B/CHMP2B and CHMP4B/CHMP2A/CHMP3 are recruited to the neck of pulled membrane tubes and reshape vesicles into helical "corkscrew-like" membrane tubes. Sub-tomogram averaging reveals that the ESCRT-III filaments assemble parallel and locally perpendicular to the tube axis, highlighting the mechanical stresses imposed by ESCRT-III. Our results underline the versatile membrane remodeling activity of ESCRT-III that may be a general feature required for cellular membrane remodeling processes

Interplay between the Conformational Dynamics of a Bacterial ABC-Transporter and Surrounding Membrane Mechanical Properties

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